Carboxylic acids are organic compounds that contain at least one carboxyl group. Carboxylic acids occur widely and include the amino acids (which make up proteins) and acetic acid (which is part of vinegar and occurs in metabolism) for instance. Carboxylic acids are used in the production of polymers, pharmaceuticals, solvents, and food additives. Industrially important carboxylic acids include acetic acid (component of vinegar, precursor to solvents and coatings), acrylic and methacrylic acids (precursors to polymers, adhesives), adipic acid (polymers), citric acid (beverages), ethylenediaminetetraacetic acid (EDTA) (chelating agent), fatty acids (coatings), maleic acid (polymers), propionic acid (food preservative), terephthalic acid (polymers), butyric acid (food additive), succinic acid (food additive, polymers).
Glycolic acid (HOCH2COOH, CAS Number: 79-14-1), or glycolate for its conjugate base, is the simplest member of the alpha-hydroxy acid family of carboxylic acids. Glycolic acid has dual functionality with both alcohol and moderately strong acid functional groups on a very small molecule. Its properties make it ideal for a broad spectrum of consumer and industrial applications, including use in water well rehabilitation, leather industry, oil and gas industry, laundry and textile industry, cleaning products, and as a component in personal care products. It can also be used to produce a variety of polymeric materials, including thermoplastic resins comprising polyglycolic acid which have excellent gas barrier properties, and thus may be used to make packaging materials having the same properties (e.g., beverage containers, etc.). The polyester polymers gradually hydrolyze in aqueous environments at controllable rates. This property makes them useful in biomedical applications such as dissolvable sutures and in applications where a controlled release of acid is needed to reduce pH. Currently, more than 50,000 tons of glycolic acid are consumed annually worldwide.
Although glycolic acid occurs naturally as a trace component in sugarcane, beets, grapes and fruits, it is mainly synthetically produced. Other technologies to produce glycolic acid are described in the literature or in patent applications. For instance, EP 2025759 and EP 2025760 patent applications describe a method for producing the said hydroxycarboxylic acid from aliphatic polyhydric alcohol having a hydroxyl group at the end by using a microorganism. This method is a bioconversion, as is the one described by Michihiko Kataoka in the paper on the production of glycolic acid using ethylene glycol-oxidizing microorganisms (Kataoka et al., 2001). Glycolic acid is also produced by bioconversion from glycolonitrile using mutant nitrilases with improved nitrilase activity and that technique was disclosed in patent applications WO 2006/069110, WO 2009/059104, and WO 2009/059096, or by bioconversion from ethylene glycol, glycolaldehyde or glyoxal as disclosed in patent applications JP 2007/228927 or WO 2005/106005. Methods and microorganisms for producing glycolic acid by fermentation from renewable resources wherein carbohydrates are converted to glycolic acid by one direct step of fermentation were disclosed in patent applications WO 2007/141316, WO 2010/108909, WO 2011/036213, WO 2011/157728, WO 2012/025780, CN105647844A, CN106011185A, and WO 2016/079440 for methods using Escherichia coli strains, and in WO 2013/050659, WO 2014/162063, and WO 2016/193540 using Saccharomyces cerevisiae or Kluyveromyces lactis strains.
Glyoxylic acid or oxoacetic acid (HCOCOOH, CAS Number: 298-12-4) or glyoxylate for its conjugate base is a C2 carboxylic acid. Glyoxylic acid is an intermediate of the glyoxylate cycle, which enables organisms, such as bacteria, fungi and plants to convert fatty acids into carbohydrates. Glyoxylate is the byproduct of the amidation process in biosynthesis of several amidated peptides. It is a colourless solid that occurs naturally and is useful industrially. It is used as a cleaning agent for a variety of industrial applications, as a specialty chemical and biodegradable copolymer feedstock and as an ingredient in cosmetics. It is a useful compound for agricultural and pharmaceutical chemicals. Indeed, glyoxylic acid can be used in pharmaceutical industry since its condensation with phenols gives 4-hydroxymandelic acid which reacts with ammonia to give hydroxyphenylglycine, a precursor to the drug amoxicillin or which can be reduced to give 4-hydroxyphenylacetic acid, a precursor to the drug atenolol. Moreover acid-catalysed reaction of glyoxylic acid with urea leads to the production of allantoin used in cosmetics, ointments and in the treatment of some cancers (Cativiela et al., 2003). Finally, condensation with guaiacol in place of phenol provides a route to vanillin, used as a flavoring agent in foods, beverages, and pharmaceuticals.
Although glyoxylic acid occurs naturally as a trace component in unripe fruit and young green leaves, it is mainly synthetically produced. Other technologies to produce glyoxylic acid are described in the literature or in patent applications. For instance, glyoxylic acid may be chemically produced by heating dibromoacetic acid with some water or by electrolytic reduction of oxalic acid or by nitric oxidation of glyoxal. Some patent applications describe processes of production of glyoxylic acid by bioconversion, such as patent applications WO 1993/14214, U.S. Pat. No. 5,439,813, and WO 1994/28155 disclosing the bioconversion from glycolic acid using glycolate oxidase produced by a microorganism, as well as Isobe & Nishise (1999). Patent application US 2007/0026510 discloses the bioconversion from glyoxal using an aldehyde oxidase.
The industrial interest of glycolic acid and glyoxylic acid coupled with environmental concerns due to chemical by-products formed during chemical productions render microbial production of such carboxylic acids an attractive prospect.
The inventors have identified new methods for the production of glycolic acid and/or glyoxylic acid from carbohydrates as sole carbon source involving at least one fermentative step and a modified microorganism in which activity of phosphoketolase is enhanced.
Phosphoketolase activity and genes encoding enzymes with such activities are known in the art (Papini et al., 2012). Two different phosphoketolase activities have been reported in bacteria. Xylulose 5-phosphate phosphoketolase catalyses the phosphate consuming conversion of xylulose 5-phosphate to glyceraldehyde 3-phosphate and acetylphosphate with release of water. Xylulose 5-phosphate phosphoketolase is, for instance, encoded by the xpkA gene from Lactobacillus pentosus (Posthuma et al., 2002). Fructose 6-phosphate phosphoketolase catalyses the phosphate consuming conversion of fructose 6-phosphate to erythrose 4-phosphate and acetylphosphate with release of water. Few genes encode phosphoketolases having both xylulose 5-phosphate phosphoketolase and fructose 6-phosphate phosphoketolase activities, as, for instance, is the case for the protein encoded by the gene xfp from Bifidobacterium lactis (Meile et al., 2001) or Bifidobacterium animalis (WO 2006/016705 and WO 2016/044713).
The use of phosphoketolase for the production of metabolites of interest is already known and has been disclosed in patent applications WO 2006/016705 and in WO 2016/044713. The metabolites of interest are glutamic acid, glutamine, proline, arginine, leucine, cysteine, succinate, polyhydrobutyrate and 1,4-butanediol. The use of phosphoketolase for the production of glycolic acid or glyoxylic acid has never been disclosed.
The methods and the microorganisms of the invention are new over the prior art since the use of phosphoketolase for the production of glycolic acid and/or glyoxylic acid has never been previously disclosed. The inventors have surprisingly found that the overproduction of phosphoketolase in the microorganisms of the invention improves the production of glycolic acid and/or glyoxylic acid.
The methods identified by the inventors involve either only one step of fermentation of the modified microorganisms of the invention or one step of fermentation of the modified microorganisms of the invention and one step of either biological or chemical conversion of the glycolic acid or of the glyoxylic acid.